Anchoring of a monotopic membrane protein: the binding of prostaglandin H2 synthase-1 to the surface of a phospholipid bilayer

Prostaglandin H₂ synthases (PGHS-1 and -2) are monotopic peripheral membrane proteins that catalyse the synthesis of prostaglandins in the arachidonate cascade. Picot et al. (1994) proposed that the enzyme is anchored to one leaflet of the bilayer by a membrane anchoring domain consisting of a right-handed spiral of amphipathic helices (residues 73–116) forming a planar motif. Two different computational approaches are used to examine the association of the PGHS-1 membrane anchoring domain with a membrane via the proposed mechanism. The electrostatic contribution to the free energy of solvation is obtained by solving numerically the finite-difference Poisson equation for the protein attached to a membrane represented as a planar slab of low dielectric. The nonpolar cavity formation and van der Waals contributions to the solvation free energy are assumed to be proportional to the water accessible surface area. Based on the optimum position determined from the continuum solvent model, two atomic models of the PGHS-1 anchoring domain associated with an explicit dimyristoylphosphatidylcholine (DMPC) bilayer differing by the thickness of the membrane bilayer were constructed. A total of 2 ns molecular dynamics simulation were performed to study the details of lipid- protein interactions at the microscopic level. In the simulations the lipid hydrocarbon chains interacting with the anchoring domain assume various shapes, suggesting that the plasticity of the membrane is significant. The hydrophobic residues in the membrane side of the helices interact with the hydrophobic membrane core, while the positively charged residues interact with the lipid polar headgroups to stabilize the anchoring of the membrane domain to the upper half of the bilayer. The phosphate headgroup of one DMPC molecule disposed at the center of the spiral formed by helices A, B, C and D interacts strongly with Arg120, a residue on helix D that has previously been identified as being important in the activity of PGHS-1. In the full enzyme structure, this position corresponds to the entrance of a long hydrophobic channel leading to the cyclooxygenase active site. These observations provide insights into the association of the arachidonic acid substrate to the cyclooxygenase active site of PGHS-1. Received: 20 December 1999 / Revised version: 26 March 2000 / Accepted: 26 March 2000     

Dynamic survey of mitochondria by ubiquitin

Ubiquitin is a post‐translational modifier with proteolytic and non‐proteolytic roles in many biological processes. At mitochondria, it performs regulatory homeostatic functions and contributes to mitochondrial quality control. Ubiquitin is essential for mitochondrial fusion, regulates mitochondria‐ER contacts, and participates in maternal mtDNA inheritance. Under stress, mitochondrial dysfunction induces ubiquitin‐dependent responses that involve mitochondrial proteome remodeling and culminate in organelle removal by mitophagy. In addition, many ubiquitin‐dependent mechanisms have been shown to regulate innate immune responses and xenophagy. Here, we review the emerging roles of ubiquitin at mitochondria.     

Host PI(3,5)P2 Activity Is Required for Plasmodium berghei Growth During Liver Stage Infection

Malaria parasites go through an obligatory liver stage before they infect erythrocytes and cause disease symptoms. In the host hepatocytes, the parasite is enclosed by a parasitophorous vacuole membrane (PVM). Here, we dissected the interaction between the Plasmodium parasite and the host cell late endocytic pathway and show that parasite growth is dependent on the phosphoinositide 5‐kinase (PIKfyve) that converts phosphatidylinositol 3‐phosphate [PI(3)P] into phosphatidylinositol 3,5‐bisphosphate [PI(3,5)P₂] in the endosomal system. We found that inhibition of PIKfyve by either pharmacological or non‐pharmacological means causes a delay in parasite growth. Moreover, we show that the PI(3,5)P₂ effector protein TRPML1 that is involved in late endocytic membrane fusion, is present in vesicles closely contacting the PVM and is necessary for parasite growth. Thus, our studies suggest that the parasite PVM is able to fuse with host late endocytic vesicles in a PI(3,5)P₂‐dependent manner, allowing the exchange of material between the host and the parasite, which is essential for successful infection.      

Trace Metal Concentration in a Temperate Freshwater Reservoir Seasonally Subjected to Blooms of Toxin-Producing Cyanobacteria

In situ interactions between cyanobacteria and metals were studied at Torrão reservoir (Tâmega River, North Portugal). The metal content of water and sediments from the reservoir was monitored monthly at Marco de Canaveses (seasonally subjected to toxic blooms of Microcystis aeruginosa) and upstream at Amarante (no blooms recorded), for 16 months. During the 16 months of the study period, M. aeruginosa bloomed twice at Marco de Canaveses, firstly forming a scum, and later with colonies scattered throughout the reservoir. Metals Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn were analysed in the sediment and in the water column. Cu-binding ligands in water were also determined. When no blooms were taking place, average metal levels for water and sediment were not statistically different at both locations. Therefore, it was considered that the absence of cyanobacteria blooms at Amarante was not due to differences in metal content. When blooms were taking place at Marco de Canaveses, a significant increase of metal levels in the sediment occurred simultaneously. Sediment quality guidelines showed that during this period, Cu and Pb concentrations (32.3 and 43.2 mg kg^?1, respectively) were potentially toxic. However, quantification of the exchangeable metal fraction indicated that these metals were probably not bioavailable. Concentration of Cu-binding ligands in water was higher during the blooms, indicating that cyanobacteria are capable of changing the metal speciation in situ in a reservoir.     

A conjugate of the lytic peptide Hecate and gallic acid: structure,activity against cervical cancer,and toxicity

Conjugate compounds constitute a new class of molecules of important biological interest mainly for the treatment of diseases such as cancer. The N-terminus region of cationic peptides has been described as important for their biological activity. The aim of this study was to evaluate the lytic peptide Hecate (FALALKALKKALKKLKKALKKAL) and the effect of conjugating this macromolecule with gallic acid (C₇H₆O₅) in terms of structure, anti-cancer activity, and toxicity. An N-terminus GA-Hecate peptide conjugate was synthesized to provide information regarding the relationship between the amino-terminal region and its charge and the secondary structure and biological activity of the peptide; and the effects of gallic acid on these parameters. Peptide secondary structure was confirmed using circular dichroism (CD). The CD measurements showed that the peptide has a high incidence of α-helical structures in the presence of SDS and LPC, while GA-Hecate presented lower incidence of α-helical structures in the same chemical environment. An evaluation of the anti-cancer activity in HeLa cancer cells indicated that both peptides are active, but that coupling gallic acid at the N-terminus decreased the activity of the free peptide. GA-Hecate showed lower activity in non-tumor keratinocyte cells but higher hemolytic activity. Our findings suggest that the N-terminus of Hecate plays an important role in its activity against cervical cancer by affecting it secondary structure, toxicity, and hemolytic activity. This study highlights the importance of the N-terminus in antitumor activity and could provide an important tool for developing new anti-cancer drugs.     

Real-time PCR method to quantify Sp245 strain of Azospirillum baldaniorum on Brachiaria grasses under field conditions

Plant and Soil - Soil microbiome roles in agriculture is becoming more and more important. This importance is also reflected on the way plants are seen: complex organisms formed by the plant itself...     

Unusual adult-onset Leigh syndrome presentation due to the mitochondrial m.9176T>C mutation

Leigh syndrome (LS) is an incurable, nearly always fatal, neurodegenerative, pediatric disorder that results from respiratory chain failure. The most common mitochondrial DNA (mtDNA) mutations that result in LS are m.8993T→C/G and m.9176T→C/G, which were previously found in several patients with early-onset Leigh syndrome. Here, we describe clinical and molecular features of a novel pedigree, where LS developed in two siblings. The proband was a young woman with an unusual adult-onset LS. She harbored a homoplasmic m.9176T→C mutation, based on analysis of a muscle biopsy. In contrast, the brother died at a young age. This novel case report and literature review highlights the variability of phenotypic expression of the m.9176T→C mutation.